Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

Summary A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.


Introduction
Whole genome sequencing (WGS) is increasingly recognized as a vital technique in (diagnostic) genetic screening of disease, including inherited retinal diseases (IRDs), such as USH2A-associated Usher syndrome type 2 (USH2, MIM: #276901) and autosomal recessive retinitis pigmentosa (arRP, MIM: #613809). The added value compared with whole exome sequencing (WES) and most targeted sequencing panels is that it detects (novel) deep-intronic variants, 1-3 structural variants (SVs), 4,5 and variants in regulatory regions, 6 resulting in an increased diagnostic yield in IRD cases. 4,7,8 In previous studies, WES and targeted sequencing panels solved 36% to 63% of persons with non-syndromic arRP and 84% to 90% of Usher syndrome (USH) cases. 4,9 We hypothesized that a substantial portion of the remaining unsolved cases has an SV or a deleterious deep-intronic variant that potentially results in inclusion of a pseudoexon (PE). To date, 80 (likely) pathogenic SVs (larger than 50 nucleotides) have already been submitted to ClinVar (March 29, 2022) and several deep-intronic variants resulting in PE inclusion have been reported for USH2A. 1-3 Therefore, it is plausible that novel SVs and deep-intronic variants that cause PE inclusion will be identified when genetically unexplained cases are screened with WGS.
The USH2A gene is one of the most frequently mutated IRD-associated genes, implicated in both USH2 and arRP, as pathogenic variants are identified in 57% to 79% and 12% to 25% of affected individuals, respectively. 10 USH2A-associated IRD is still considered untreatable. However, several gene-specific treatment options are currently in different stages of (pre-)clinical development, including splicemodulation using antisense oligonucleotides (AONs), 11 CRISPR-Cas9-based gene correction, 12 and exon-excision. 13 Ultevursen, an AON that induces the skipping of USH2A exon 13, is currently being evaluated in a phase II/III clinical trial (ClinicalTrials.gov Identifier: NCT05158296). These developments further stress the importance for subjects to obtain a conclusive genetic diagnosis in order to be eligible for future personalized therapies.
In this study, we aimed to identify missing variants in USH2A in a cohort of 100 largely mono-allelic arRP and USH2 subjects through WGS, assess an effect on splicing of all detected single nucleotide variants (SNVs), and to develop an AON-based splice correction strategy for specific deep-intronic variants. In addition, our approach could serve as a future framework for detection and treatment of variants in genes associated with other hereditary disorders.

Ethics declaration
Written informed consent was obtained by the corresponding centers, adherent to the tenets of the declaration of Helsinki and as approved by the local ethics committee of the Radboud University Medical Center Nijmegen, as an amendment to the approval by the local ethics committee of the Rotterdam Eye Hospital (MEC-2010-359;OZR protocol no. 2009-32).
Whole genome sequencing DNA of unrelated probands was collected from subjects from the Netherlands, Germany, Ireland, Northern Ireland, Israel, Russia, Czech Republic, Switzerland, France, Italy, Denmark, Canada, and Belgium. Subjects were included if they were clinically diagnosed with arRP (n ¼ 47), USH2 (n ¼ 49), or autosomal recessive hearing impairment (DFNB, n ¼ 3) in those who were still in the first or second decade of life. One subject with cone-rod dystrophy (CRD) and hearing loss was included. In addition, we required that pre-screening of the (majority of) USH2A exons was performed and only one (potentially or likely) pathogenic variant in USH2A was identified or no USH2A variants were identified. In 67 subjects, a (potentially or likely) pathogenic variant in USH2A was reported prior to WGS screening (34 for RP, 30 for USH, and three for DFNB). USH2A variants that were identified in probands prior to their inclusion in our WGS analysis pipeline are shown in Table 1 and screening methods that were used prior to WGS are listed in Table S1.
WGS was performed as previously described. 8,18 Variants in the entire USH2A gene (chr1:215796236-216596738), 10 previously determined potential regulatory regions of USH2A, 6 the known retinal modifier of USH2A-associated disease PDZD7, 19 and variants in the entire genomic regions of other genes associated with USH (n ¼ 17, Table S2; RetNet; visited May 21, 2021) and arRP (n ¼ 62, Table S2) were extracted and evaluated. Variants with a gnomAD allele frequency (AF) % 1% in the general population and in any subpopulation were prioritized. SNVs were considered potentially pathogenic if they were (1) a stop gain variant, frameshift variant, in-frame insertion, or deletion or a canonical splice site variant; (2) a missense variant with a Grantham score R80 (range: 5-215), CADD_PHRED R15 (range: 0-99) or PhyloP R2.7; or (3) a putative splice-modulating variant with two out of four SpliceAI D scores R0.1 or one SpliceAI D score R0.15 (default settings, range 0-1). 20 Copy number variants (CNVs) were called with control-FREEC and SVs were called with Manta Structural Variant Caller V.1.1.0. 21,22 CNVs and SVs were prioritized if they passed the internal quality filter as previously described 8 ; had a high quality score (R100 of 1,000); had an AF % 1% in databases, such as gnomAD, Decipher, and GoNL; and affected at least one exon of USH2A or the aforementioned 62 arRP-associated or 17 USH-associated genes. Segregation analysis was performed if DNA samples of relatives were available.

Minigene splice assays
Variants predicted to have an effect on splicing (two out of four SpliceAI D scores R0.1 or one SpliceAI D score R0.15) were selected for a minigene splice assay. Four variants were selected prior to the release of SpliceAI and did not meet these criteria. Two of these variants (c.1644þ7453A>G and c.4396þ6885T>C) were included because an effect on pre-mRNA splicing was predicted by splice prediction tools incorporated in Alamut Visual Plus. Variant c.8710G>A was included due to its 1 base pair downstream location of c.8709C>T that was shown to have an effect on pre-mRNA splicing in a previous study, 9 whereas a fourth variant (c.6806-7599C>G) was selected as a unique variant absent from gnomAD in one of the first samples that was analyzed with  WGS. Selected variants were either deep-intronic variants, non-canonical splice site variants, missense variants, or variants that affected a ''branchpoint A'' upstream of an exon. Constructs were generated with Gateway cloning technology (Thermo Fisher Scientific, Carlsbad, CA, USA) and minigene splice assays were performed as published previously. 23 Primer sequences are listed in Table S3.

Variant classification
SNVs were classified according to the American College of Medical Genetics (ACMG) guidelines with Franklin and SVs were manually classified according to published ACMG guidelines. 24,25 SNVs with an effect on pre-mRNA splicing were classified based on their effect in the minigene splice assay; variants with a full stop gain effect in all transcripts were classified as pathogenic, variants resulting in an in-frame deletion thereby disrupting a predicted functional protein domain were classified as likely pathogenic and variants with remaining conventionally spliced transcript were classified as variant of uncertain significance (VUS). Subjects with two (likely) pathogenic variants were considered genetically solved, subjects with one (likely) pathogenic variant and one VUS were considered ''possibly solved.''

Antisense oligonucleotides
AONs were designed according to previously published guidelines 26,27 and purchased from Eurogentec (Seraing, Belgium) with 2 0 -O-MOE modifications and a complete phosphorothiorate backbone. For each PE, at least two AONs were designed complementary to splice acceptor or donor sites, or exonic splice enhancer regions. Only one AON targeting the PE resulting from c.4885þ375A>G could be designed due to low GC-content of the PE sequence. NCBI blast searches were performed to exclude potential off-target binding sites for each designed AON. Moreover, a scrambled oligonucleotide (SON) as well as an AON containing three mismatches compared with the most potent AON for each target (3ntMM AON) were designed and purchased. All AONs were dissolved in sterile phosphate buffered saline (PBS 1x) to a stock concentration of 1 mM and diluted in PBS to concentrations of 0.01 mM, 0.02 mM, 0.05 mM, and 0.1 mM. One microliter of each AON was co-transfected in duplicate with individual minigene splice assay plasmids containing the variant of interest, as described in the minigene splices assays section. Transfected cells were subjected to RNA analyses as described before. 23

Generation of photoreceptor precursor cells
Blood was obtained from subjects arRP36 (heterozygous for c.4397-3890A>G), USH46 (heterozygous for c.1551-504C>T), and a healthy control. Peripheral blood mononuclear cells were isolated and reprogrammed into induced pluripotent stem cells (iPSCs) through episomal nucleofection, as described previously, 28 and subsequently differentiated into photoreceptor precursor cells (PPCs). 17,29 iPSC lines were generated at the Stem Cell Technology Center of the Radboudumc (Nijmegen, the Netherlands). AONs or a SON were gymnotically delivered to the PPCs on day 28 by adding them directly to the culture medium. Cycloheximide (CHX, in a final concentration of 100 mg/mL) was added on day 29 and cells were harvested in sterile PBS on day 30. Levels of PE inclusion and reversal after treatment were determined with PCR and RT-qPCR (in duplicate) according to standard protocols (primer sequences in Table S4). AON efficacy was assessed based on the splicing correction capacity at the different AON concentrations.

Results
Forty-one probands were (likely) solved having biallelic USH2A variants One hundred unrelated subjects were included for WGS analysis. Forty-seven of these subjects were clinically diagnosed with non-syndromic arRP, 49 with USH, three with DFNB, and one with CRD and hearing loss. The subjects with DFNB had one (likely) pathogenic USH2A variant and were still in the first or second decade of life, potentially not (yet) presenting early signs of vision loss. In total, 6,054 unique SNVs were identified within USH2A and predicted regulatory elements. Of these, 2,820 variants had a gnomAD AF %1% in the general population. Ninety-seven variants were considered potentially pathogenic, as they met our inclusion criteria for being a stop gain variant, frameshift variant, in-frame insertion or deletion, a canonical splice site variant, or a potentially pathogenic missense variant or putative splice-modulating variant (Table S5). Nineteen of these SNVs were not reported in ClinVar or the Leiden Open (source) Variation Database (LOVD) and were therefore considered novel variants in our study. Twelve USH2A SVs on 13 alleles adhered to our criteria, including two duplications, two inversions, 16 and eight deletions of which one (c.4627þ25436_4987þ659del) was identified in two probands. The largest SV with both breakpoints within USH2A was a duplication from exon 15 to 65 (c.2994-3030_14343þ488dup), the shortest SV was a deletion of exon 43 (c.8655_8681þ1681del). All SVs were confirmed with PCR and subsequent Sanger sequencing. After assessment of all individual variants from the total cohort, 41 cases were genetically explained with (likely) biallelic USH2A variants. An overview of all variants that were classified as (likely) pathogenic or were VUS that were deemed potentially causative is presented in Table 1.
All samples were also evaluated for the presence of potentially pathogenic variants in other arRP-or USH-associated genes (Table S2), applying the same criteria to detect biallelic variants in these genes. We identified 133 unique variants in these genes, resulting in eight of the remaining probands being (possibly) solved with biallelic variants in the following genes: ARSG (two cases, described in Velde et al. 14 ), EYS, MYO7A, PEX6, PQCL2 (described in Millo et al. 15 ), PROM1, and RPE65 (described in Panneman et al. 30 ). An overview of solve rates is visualized in Figure 1.
Assessment of the effect of variants on splicing using minigene splice assays We identified 36 variants with two out of four SpliceAI D scores R0.1 or one SpliceAI D score R0.15 (Table S6). 20 Stop gain variants (n ¼ 2) and canonical splice site variants (n ¼ 7) were excluded from these analyses as they are generally considered to be pathogenic. For three variants (c.5573-834A>G, c.7595-2144A>G, and c.12295-3T>A) a deleterious effect on USH2A pre-mRNA splicing was already previously determined 1,2,31 and they were therefore omitted for testing. We also excluded one variant that was an artifact and confirmed to be absent with Sanger sequencing (c.1551-508C>T), one variant in a proband that would remain mono-allelic after performance of a minigene splice assay (c.4732C>T), and one variant for which gain of canonical splice sites was predicted (c.9975G>A) (Table S6). Minigene splice assays were performed for the remaining 21 variants (Figures 2A-2D, Table S7). Thirteen of these variants showed a(n) (partial) effect on splicing ( Figure S1), of which eight variants affected pre-mRNA splicing in all amplified transcripts and these were therefore classified as (likely) pathogenic. The remaining five variants (c.1841-377A>G, c.2303G>A, c.9959-3C>G, c.14134-5T>C, and c.14583-26A>G) only revealed a partial effect on splicing and were therefore classified as VUS.
(a) Deep-intronic variants (n ¼ 7) Seven of the 21 variants that were evaluated in a minigene splice assay were deep-intronic USH2A variants (Figure 2A).  Design and evaluation of antisense oligonucleotides to correct aberrant pre-mRNA splicing events AONs and controls were designed and evaluated for their potential to correct aberrant splicing induced by four deep-intronic variants resulting in PE inclusion (c.1551-504C>T, c.1841-377A>G, c.4397-3890A>G, and c.4885þ 375A>G). Characteristics of all AONs and their position relative to the targeted PE and exonic splice enhancers are described in Table S8 and Figure S2.
For all four targets, at least one of the AONs was capable of redirecting aberrant splicing at the lowest concentration (0.01 mM) using the matching minigene splice assay vectors ( Figure 3). Transfection with an SON did not show effect for three of the four targets, which indicates that the splicing correction is sequence-specific. To further confirm the sequence-specificity, 3ntMM AONs were also assessed for their potential to redirect splicing in the corresponding minigene splice assays. For c.1551-504C>T and c.4397-3890A>G, these 3ntMM AONs were indeed less effective than the original AONs, even at the highest concentration used (0.1 mM), corroborating the effect of the original AON to be sequence-specific. In contrast, no difference in efficiency of splicing redirection was observed between the original and 3ntMM AONs against c.1841-377A>G (PE10) and c.4885þ375A>G (PE23) at all tested concentrations. The SON of PE23 also redirected splicing. Therefore, it is unclear whether or not the effect of the designed AON to correct aberrant PE23 splicing is sequence-specific.
AONs are effective in patient-derived PPCs PPCs were generated from iPSCs derived from two subjects (arRP36 and USH46) who were confirmed compound heterozygous for c.4397-3890A>G (PE20) and c.2276G>T (p.(Cys759Phe)) and c.1551-504C>T (PE8) and c.9959-3C>G (p.[Met3321Asnfs*22,¼,Gly3320_Ser3338del]), respectively. These PPCs enabled us to confirm the PE inclusion caused by these variants and to test the AONs in a cellular model resembling human photoreceptors within the entire genetic context of the patient. RT-qPCR analyses of several neuronal progenitor, photoreceptor, and retinal pigment epithelium markers indicated that the differentiation of iPSCs into PPCs was successful for both cell lines ( Figure S3).
As expected, PE inclusion was observed in PPCs derived from subjects having the c.1551-504C>T (PE8) or c.4397-3890A>G (PE20) variant ( Figure 4). For variant c.4397-3890A>G, AON2 was effective in redirecting aberrant splicing at all three tested concentrations, while the 3ntMM AON and SON did not show any effect. This effect was confirmed with RT-qPCR as a reduction of 49% (after treatment with 0.01 mM AON) to 92% (treated with 0.1 mM AON) of transcripts containing PE20 as compared with the control sample (treated with CHX, but not with AON) was observed. Interestingly, inclusion of an additional PE (PE20a) was observed in the untreated samples and samples that were treated with 3ntMM AON and SON, but not in the samples treated with AON2. This PE20a is possibly a result of variant c.4396þ7263del and is assumed to be part of the c.4397-3890A>G haplotype because it was detected with WGS in both subjects having the c.4397-3890A>G variant.
For variant c.1551-504C>T, AON3 redirected aberrant USH2A pre-mRNA splicing, albeit with a lower efficiency than was observed in the minigene splice assay, as still remaining PE8-containing transcripts could be detected after AON treatment in all tested concentrations. Compared with the amount of transcript that contains PE8 in the control sample that was not treated with AON, a reduction of 33% (treated with 0.1 mM AON) to 50% (treated with 1 mM AON) of PE8-containing transcript was observed.

Discussion
Through our study, 49 out of 100 (49%) previously unsolved arRP and (suspected) USH individuals now received a conclusive or possibly conclusive genetic diagnosis. Our analysis confirmed that WGS is a powerful method for solving cases that remained genetically unsolved after screening the USH2A exons and flanking intronic regions. Using the combination of WGS and subsequent dedicated minigene splice assays, we identified novel pathogenic deep-intronic variants and SVs and confirmed a splice defect for other exonic, branchpoint and non-canonical splice site variants.
WGS followed by testing of splice defects should therefore be considered as a valuable method to increase genetic solve rates, which is especially important in the current era of upcoming genetic therapies and increased focus on personalized medicine. Furthermore, we showed that AONs are a valuable tool to correct aberrant splicing as a consequence of deep-intronic variants in USH2A.
To compare the efficacy of our WGS analysis with published WGS studies is complex, due to varying prescreening conditions and inclusion criteria. However, the solve rate in our study seems in line with other WGS studies that include USH2A variants. A WGS study from the United Kingdom solved 31% of persons with IRDs that did not receive a complete genetic diagnosis after WES and 55% of cases that were directly screened with WGS. 4 In another study in which 57 mono-allelic IRD cases from the Netherlands, Ireland, and Israel were included that were previously screened with WES or target-capture sequencing, the solve rate was 21% (12 of 57). 8 In our study, we solved 49% of cases that underwent prior testing using WES or targeted sequencing, such as molecular inversion probe-based sequencing. We identified several variants that are not (easily) being detected by WES or targeted sequencing, such as pathogenic deep-intronic USH2A variants (in seven cases) or SVs (13 alleles in 12 cases). SVs have been previously shown to explain a portion of missing heritability in cases that were screened with targeted sequencing panels. 33 The detection of 13 (likely) pathogenic USH2A SVs in our cohort of 100 subjects, representing 11% of (likely) pathogenic variants identified within USH2A, emphasized the value of WGS compared with targeted sequencing panels and WES for a substantial increase in diagnostic yield for IRDs.
Of the solved probands, 22 subjects had SNVs that could have been detected through WES or targeted sequencing. This was likely to be partially caused by lack of detection due to incomplete screening with targeted sequencing. However, nine variants resulted in deviations in pre-mRNA splicing in a minigene splice assay and were located either in an exon or in a non-canonical splice site region. Previous reports estimated that up to a third of disease-causing variants disrupt pre-mRNA splicing, either directly or through mis-regulation of splice-regulating factors. 34 Our results emphasize that increased knowledge on variant interpretation and functional testing of possible (B) In PPCs heterozygous for c.4397-3890A>G, inclusion of PE20 was fully reverted after treatment with AON2 against PE20, while an SON and 3ntMM AON did not have an effect. (C and D) The effect observed with RT-PCR was confirmed with RT-qPCR. Levels of transcripts containing the PE of interest as well as WT transcript was compared with USH2A exon 13 expression (normalized against GUSB) and confirmed that AON3 against PE8 can partially rescue PE8 inclusion, while AON2 against PE20 has a full effect. (E and F) Expression of USH2A (exon 13) compared with GUSB. USH2A expression was relatively low in all samples; however, lowest in samples not treated with cycloheximide (CHX). effects on splicing of branchpoint variants, missense variants, synonymous variants, and non-canonical splice site variants will increase diagnostic yield in USH2A screening, and in genetic testing for hereditary disorders in general. Re-evaluation of targeted sequencing and WES data of currently still unsolved cases with a particular focus on splice-modulating properties is therefore recommended.
The genetic diagnosis for 13 subjects could be completed after performing a minigene splice assay for variants that were selected based on our criteria for SpliceAI. Eight of these variants displayed an effect on all detected transcripts in the corresponding minigene splice assays ( Figure S1 and Table S7). To our knowledge, the c.5573-19A>G variant, reported previously as VUS in both LOVD and ClinVar, is one of the first suggested branchpoint variants within USH2A resulting in a pathogenic effect. 35 The identification of an additional potential branchpoint variant c.14583-26A>G (p.[¼,Tyr4862Alafs*22]) stresses the importance of including the evaluation of branchpoint variants in genetic screening efforts. Five variants that were tested in a minigene splice assay showed residual conventionally spliced transcripts along with a deleterious effect on splicing and they were therefore classified as VUS.
Recent studies have been published on specificity and sensitivity of splice prediction tools. 36,37 In a study by Rowlands et al., 36 SpliceAI (suggested D score R0.2) outperformed other splice prediction tools, although false positive and false negative calls were observed. In general, our findings are in line with the findings of Rowlands et al. that SpliceAI is able to adequately predict effects on pre-mRNA splicing. Only four of the 17 variants (24%) that met our inclusion criteria (two out of four SpliceAI D scores R0.1 or one D score R0.15) did not display an effect on pre-mRNA splicing in HEK293T cells, which were all exonic variants. Four other variants, including one exonic variant, that were tested in this study did not meet our SpliceAI inclusion criteria, but for none of these variants an effect on pre-mRNA splicing was observed. The inclusion criteria in our study were less restrictive compared with those used by Rowlands et al. and Riepe et al. (R0.2), 36,37 yielding three additional variants (c.1841-377A>G, c.5573-19A>G, and c.5775A>T) with a maximum SpliceAI D score between 0.1 and 0.2, which did show an effect on pre-mRNA splicing. We would therefore suggest using 0.1 as a criterion for SpliceAI for deep-intronic, non-canonical splice site and branchpoint variants, but this could be adapted for exonic variants as no effect on pre-mRNA splicing was observed for the majority of exonic variants that we tested. A SpliceAI criterion of R0.2 for exonic variants outside the non-canonical splice site regions would exclude all exonic variants that did not have an effect on pre-mRNA splicing in our minigene splice assays but would yield the remaining variant (c.2303G>A) that did have an effect. The use of other splice prediction tools in addition to SpliceAI could also be considered for this type of variants.
Screening of variants with a potential effect on pre-mRNA splicing in a broader genetic context, such as larger minigenes or use of patient-derived PPCs, may result in alternative outcomes. In addition, previous research has shown that recognition of splice sites can be cell type specific, 38 and the splicing patterns observed in our minigene splice assay may deviate from the natural situation in the human retina and inner ear. However, these variants have effect on USH2A pre-mRNA splicing and were all identified in arRP and USH subjects with a previously identified mono-allelic pathogenic USH2A variant, suggesting that additional functional evidence may determine that these variants are indeed (likely) pathogenic according to ACMG guidelines. Furthermore, similar studies performed for another IRD gene, ABCA4, have shown that variants creating new splice sites are recapitulated in patientderived cells, and only those creating splicing enhancers may show tissue-dependent specificity. 38 Another important point is that little is known about the minimal amount of remaining wild-type transcript that would be required to retain normal retinal function. Functional data derived from the in-frame skipping of zebrafish ush2a exon 13 suggests that as little as 20% of functional transcripts could be enough to retain retinal function. 11 Future research in cellular and animal models will shed light on this question, potentially resulting in the reclassification of variants with an incomplete effect on splicing.
We were able to obtain patient material from two individuals carrying deep-intronic variants that was subsequently used to generate iPSCs to differentiate them to PPCs. Using these cells we could validate our findings in the minigene system. Unexpectedly, we identified an additional PE (PE20a; chr1:216356236-216356337), incorporated in the mRNA derived from cultured PPCs of subject arRP36 with variant c.4397-3890A>G (PE20). PE20a was only identified in samples that were treated with CHX, and treatment with AON against PE20 also appeared to revert inclusion of PE20a. The presence of an additional PE could be an artifact of our assay; however, this stresses the importance of considering an entire haplotype when designing a therapeutic strategy and the importance of using large sequence parts flanking the variant of interest, preferably including flanking exons, in dedicated minigene splice assays.
We also identified variants and splicing defects in USH2A exon 13. Currently a phase II/III clinical trial to induce exon 13 skipping is ongoing. Therefore, this information is very valuable for patients, as they might be eligible to receive future treatments. Although mutations in exon 13 are relatively recurrent, this is also important for individuals with ultra-rare or even unique pathogenic variants. The impact of Milasen, an AON customized for a single subject suffering from Batten disease, has led to (inter)national initiatives to develop treatments for those ultra-rare cases with no commercial interest. 39 Highlighting once again the importance of having a concise genetic diagnosis in order to become eligible for future therapeutic genetic interventions.
Unfortunately, we were unable to provide 51 of 100 probands a solid genetic diagnosis. In most of these cases, one (likely) pathogenic USH2A variant is present, and therefore another pathogenic USH2A variant might well reside in trans. However, in the general population, approximately 36% of individuals are healthy carriers of a pathogenic variant in an IRD-associated gene. 40 While we screened for causative coding and non-coding variants in other genes associated with arRP or USH, by chance, subjects in our cohort may have a pathogenic variant in USH2A, with the underlying genetic cause of their disease elsewhere. In fact, eight cases in our cohort were solved after identification of biallelic variants in IRD genes other than USH2A. Furthermore, SVs could remain undetected or unrecognized, due to the nature of short-read sequencing techniques. 16,41,42 Pathogenic variants may also reside in low coverage regions, in genes associated with disorders that are phenotypically overlapping, or in genes not yet associated with IRDs. We did not observe potentially pathogenic variants in 10 potential regulatory regions of the USH2A gene 6 nor in the known retinal disease modifier PDZD7. 19 Improved knowledge on USH2A regulatory elements, potential modifiers, being either protective or negative, or potentially digenic inheritance patterns is likely to result in more precise classification of variants in the future.
In conclusion, we confirmed that our strategy, with elaborate WGS screening and thorough variant interpretation and thorough assessment of splice defects associated with all types of SNVs, is an effective method to significantly improve diagnostic solve rates. As a result of our strategy, we (possibly) solved 49% (49 of 100) of genetically unexplained probands with suspected USH2A-associated disease. In 21 subjects, we identified (likely) pathogenic variants in USH2A exon 13 of whom 14 subjects received a definite genetic diagnosis and these are now considered eligible for receiving Ultevursen/QR-421a-mediated exon 13 skipping therapy when available. 11 Moreover, we identified three novel pathogenic deep-intronic variants that result in the inclusion of a PE and we were able to develop effective AONs that redirected aberrant pre-mRNA splicing. Collectively, our experimental approach has been effective in the identification of novel, potentially treatable, diseasecausing variants in USH2A and can be applied to the diagnostic pipelines for all inherited disorders known so far.

Data and code availability
Data are available upon request. Pathogenic variant data are uploaded to the Leiden Open (source) Variation Database (LOVD: https://databases.lovd.nl/shared/genes/USH2A). All other whole genome sequencing data are subject to controlled access because these may compromise research participant privacy. These data may become available upon a data transfer agreement approved by the local ethics committee and can be obtained from corresponding author S.R. upon reasonable request.

Supplemental information
Supplemental information can be found online at https://doi.org/ 10.1016/j.xhgg.2023.100181.   Table S5 and Table S6 are separate excel files Figure S1: Effects on pre-mRNA splicing observed in minigene splice assays for thirteen variants. Thirteen of the 21 tested variants revealed differences in pre-mRNA splicing between mutant (M) and wildtype (WT) constructs in our minigene splice assays. The effects observed after RT-PCR are shown as well as a schematic representation of these effects including the RHO exons 3 and 5 that are flanking the USH2A genomic region cloned in the minigene. Splice predictions were obtained with SpliceAI. nt: nucleotides PE: pseudoexon Figure S2: Position of antisense oligonucleotides (AONs) and 3 nucleotide mismatch AONs (3ntMM AONs) relative to their targets. AONs were designed complementary to the splice sites or exonic splice enhancers of each specific pseudoexon (PE). The sequence of each AON is shown in green, 3ntMM AONs are in black with mismatches in red. The PE sequences that are incorporated in the mature USH2A transcript as a consequence of the identified deep-intronic variants are depicted in black, intronic sequences are in grey and the identified genetic variants are indicated in red. The bars above the sequences represent different types of putative exonic splice enhancers based on predictions from the ESEfinder tool 1 accesssed from Alamut Visual Plus v1.4.

Figure S3: Differentiation efficiency of patient-derived photoreceptor precursor cells (PPCs).
Expression of several neuronal progenitor, photoreceptor and retinal pigment epithelium markers (CRX, OPN1SW, OTX2, PAX6, RCVRN, RPE65, VDM2) as well as expression of USH2A and a pluripotency marker (NANOG) were determined with RT-qPCR in induced pluripotent stem cells (iPSCs) and compared PPCs after 30 days of differentiation. Expression was normalized against GUSB and compared to the corresponding iPSC line at the start of differentiation (day 0) using "delta delta Ct". As expected, pluripotency markers decreased while most of the other markers showed an increased. These data indicate that the differentiation of iPSCs towards PPCs was successful for both cell lines.